Biodegradation of renewable hydrocarbon fuel blends

ABSTRACT

Biologically-produced isobutanol as a component in fuel compositions provides a valuable mechanism for introducing renewable components to fuel compositions and, at the same time, provides for reduced environmental impact of the fuel composition if it were to contaminate a given environmental area.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority of U.S. Provisional Application No. 61/347,127, filed May 21, 2010, the entirety of which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of renewable fuel compositions and the fate of said compositions in the environment.

BACKGROUND OF THE INVENTION

Isobutanol is attractive as a biofuel molecule suitable for use in gasoline because it can be produced from renewable feedstocks and has many properties that potentially make it a more attractive fuel additive than ethanol; it has a greater energy density, lower water absorption, better blending ability, and it can be used in conventional combustion engines without modification (Durre, 2007, Biotech. J. 2:1525-1578). Evaluation of the effect of ethanol on the environmental fate of gasoline components has demonstrated that in aerobic systems fuel ethanol is preferentially biodegraded before the benzene, toluene, ethylbenzene, and xylene (BTEX) components of gasoline, and that ethanol degradation in aquifers rapidly consumed dissolved oxygen and other nutrients (Corseuil et al., 1998, Wat. Res. 32:2065-2072; Da Silva and Alvarez, 2002, Appl. Environ. Microbiol. 70:4720-4726; Capiro et al., 2007, Water Research, 41: 656-664). As a result, the presence of ethanol in gasoline could result in a lag in BTEX degradation and ultimately result in enhanced BTEX plumes (Powers et al., 2001, Environ. Sci. Technol. 35: 24A-30A.). Model simulations by Deeb et al (2002, J. Environ. Engin. 128: 868-875.) predicted a 16-34% increase in benzene plume lengths in the presence of ethanol, while others have suggested that the lifespan of benzene plumes may decrease with greater ethanol concentration in gasoline if greater microbial biomass is produced by growth on ethanol (Gomez and Alvarez, 2009, Water Resour. Res. 45).

A study by Mariano and colleagues (2009, Biomass and Bioenergy, 33: 1175-1181.) used indirect measurements (CO₂ evolution and dye reduction) to evaluate the affect of n-butanol on gasoline biodegradation in aerobic microcosms containing uncontaminated soil, river water, or a combination of uncontaminated soil and river water. Results of the study suggested that n-butanol may enhance gasoline degradation in soil, and that the enhancement factor may be greater than that achieved with ethanol. Results obtained by Garcia-Rivero et al. (2007, J. Environ. Eng. Sci. 6: 389-395.) also suggest that addition of n-butanol to hydrocarbon mixtures may enhance the rate of hydrocarbon aerobic biodegradation. Studies performed in the 1970s used biological oxygen demand (BOD)-based measurements to evaluate isobutanol biodegradation (Price et al 1974, J. Water Pollut. Contr. Fed. 46, 63-77; Dias and Alexander, 1971, Appl. Microbiol. 22: 1114-1118), and several studies (c.f., Deeb et al., 2000, Biodegradation, 11; 171-185; Pruden and Suidan, 2004, Biodegradation, 15:213-227; Somsamak et al., 2005, Environ. Sci. Technol. 39: 103-109; Vainberg et al., 2002, J. Environ. Eng. 128: 842-851) have evaluated the biodegradation of tert-butyl alcohol (TBA) which is the primary biodegradation product of the gasoline oxygenate methyl tert-butyl ether (MTBE) (Hatzinger et al., 2001, Appl. Environ Microbial. 67: 5601-5607; Steffan et al., 1997, Appl. Environ. Microbiol. 63: 4216-4222).

There is a need to increase renewable components of gasoline and/or transport fuel compositions without a resulting adverse environmental effect associated with the environmental fate of said compositions in circumstances of environmental contamination.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods and compositions for improving the environmental fate of hydrocarbon fuel compositions under circumstances of environmental release while increasing the renewability of said fuel compositions.

An aspect of the invention is a method for improving the environmental fate of hydrocarbon fuel compositions by the inclusion of isobutanol to said compositions resulting in improved biodegradability of one or more BTEX compounds of the gasoline. In one aspect, the methods and compositions provide improved biodegradation under anaerobic conditions. In one aspect, the methods and compositions provide improved biodegradation under aerobic conditions. In one aspect, the methods and compositions provide improved biodegradation under nitrate reducing conditions. In one aspect, the inclusion of isobutanol in hydrocarbon fuel compositions improves the biodegradation of benzene.

Another aspect of the invention is a method for improving the environmental fate of liquid fuel compositions comprising ethanol by the addition of isobutanol to said compositions resulting in improved biodegradation of one or more BTEX compounds of the gasoline.

Another aspect of the invention is a method to reduce the transport of ethanol in a soil matrix when said ethanol is a component of a hydrocarbon fuel composition released into an environmental compartment (e.g. soil, sediments, groundwater), said method comprising combining isobutanol with the fuel composition.

Another aspect of the invention is a method of reducing a BTEX plume caused by release of a hydrocarbon composition optionally comprising ethanol into an environmental compartment, said method comprising adding a suitable amount of isobutanol wherein said isobutanol acts as a cosolvent for the hydrocarbon and ethanol components of the hydrocarbon composition, thereby retarding and/or partially containing the BTEX plume and reducing the potential for its leakage into a water table.

Another aspect of the invention is directed to liquid fuel compositions comprising hydrocarbons, ethanol and isobutanol in an amount sufficient to improve the renewability of the hydrocarbon composition without increasing potential environmental impact of said composition if it were to contaminate an environmental compartment. In another aspect the renewability of the fuel composition is increased and the potential environmental impact is decreased by the inclusion of isobutanol, for example, BTEX plume expansion may be decreased as compared to a BTEX plume expansion of the same composition without the isobutanol present, particularly under aerobic environmental conditions.

Also provided herein are methods for improving the environmental fate of a hydrocarbon fuel composition comprising isobutanol in an environmental compartment under anaerobic conditions, the methods comprising adding an electron acceptor to said compartment in an amount sufficient to increase the rate of biodegradation of one or more BTEX components.

Provided herein are methods for increasing the renewability of a hydrocarbon fuel composition and limiting the impact on an environmental compartment upon contamination by said hydrocarbon fuel composition, comprising combining said hydrocarbon fuel composition with a suitable amount of isobutanol. In embodiments, the hydrocarbon fuel composition further comprises ethanol. In embodiments, the ethanol comprises up to about 10% of the fuel composition prior to addition of isobutanol. In embodiments, the isobutanol provides for improved biodegradation of at least one of the BTEX components of the hydrocarbon fuel composition. In embodiments, the isobutanol provides for improved biodegradation of benzene. In embodiments, the environmental compartment includes a soil matrix and wherein the isobutanol reduces the transport of ethanol in a soil matrix. In embodiments, the isobutanol impedes expansion of a BTEX plume from said composition. In embodiments, the isobutanol is present in an amount suitable for increasing the biodegradability of the hydrocarbon fuel composition. In embodiments, the improved biodegradation occurs under aerobic conditions. In embodiments, the improved biodegradation occurs under nitrate-reducing or sulfate-reducing conditions.

Also provided are methods of improving the environmental fate of a hydrocarbon fuel composition comprising isobutanol in an environmental compartment under anaerobic conditions comprising adding an electron acceptor to said compartment in an amount sufficient to increase the rate of biodegradation of one or more BTEX components. In embodiments, the electron acceptor is iron, sulfate, or nitrate, or a combination thereof. In embodiments, the electron acceptor is Fe(OH)₃. In embodiments, the electron acceptor is NaNO₃. In embodiments, the electron acceptor is MgSO₄. In embodiments, the one or more BTEX components comprise toluene, xylene, or benzene. In embodiments, the electron acceptor is nitrate and is added in an amount sufficient to create nitrate-reducing conditions. In embodiments, the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions. In embodiments, the electron acceptor is nitrate and is present in an amount sufficient to create nitrate-reducing conditions and wherein toluene biodegrades in about the same number of days as isobutanol. In embodiments, the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein toluene biodegrades in about the same number of days as isobutanol. In embodiments, the electron acceptor is nitrate and is present in an amount sufficient to create nitrate-reducing conditions and wherein benzene biodegrades in about the same number of days as isobutanol. In embodiments, the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation without sulfate-reducing conditions. In embodiments, the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation in the absence of isobutanol. In embodiments, the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation in the presence of ethanol.

Also provided herein are compositions comprising gasoline, isobutanol and at least one of Fe(OH)₃, NaNO₃, or MgSO₄. Also provided are compositions comprising gasoline, isobutanol and at least one of Fe(OH)₃, NaNO₃, KNO₃, NHNO₃, Na₂SO₄, CaSO₄, MgSO₄, chelated iron, zero-valent iron, and nano zero-valent iron.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 a shows isobutanol degradation up to 10 days. Error bars represent 95% confidence intervals.

FIG. 1 b shows isobutanol degradation up to 50 days.

FIG. 1 c shows ethanol degradation up to 50 days.

FIGS. 2A, B, C, and D show the impact of isobutanol on biodegradation of BTEX at high concentrations. (FIG. 2A shows benzene; 2B shows toluene; 2C shows ethylbenzene; 2D shows total xylenes.) Error bars represent 95% confidence intervals.

FIGS. 3A, B, C and D compare the impact of isobutanol and ethanol concentration on BTEX biodegradation at various treatment levels. (FIG. 3A shows benzene; 3B shows toluene; 3C shows ethylbenzene; 3D shows total xylenes.) Error bars represent 95% confidence intervals.

FIGS. 4A, B, C, and D show the impact of isobutanol on biodegradation of BTEX at lower concentrations. (FIG. 4A shows benzene; 4B shows toluene; 4C shows ethylbenzene; 4D shows total xylenes.) Error bars represent 95% confidence intervals.

FIGS. 5A, B, C, and D show isobutanol biodegradation—higher concentration—under various anaerobic reducing conditions. (FIG. 5A shows Treatment 2—Unamended; 5B shows Treatment 6—Nitrate Reducing; 5C shows Treatment 9—Iron reducing; 5D shows Treatment 12—Sulfate Reducing.) Error bars represent 95% confidence intervals. The dashed line for Treatment 2 indicates the time when the microcosms were re-amended with isobutanol.

FIGS. 6A and 6B show ethanol biodegradation in Treatment 4 (Unamended) and Treatment 14 (Sulfate reducing), respectively. Error bars represent 95% confidence intervals. The dashed line for Treatment 4 indicates the time when the microcosms were re-amended with ethanol after the residual sulfate in the groundwater was reduced.

FIGS. 7A, B, C, and D show benzene and toluene biodegradation (higher concentration) under various anaerobic reducing conditions and in the presence or absence of isobutanol (IBA). (A and B show benzene and toluene, respectively, for Treatments 1, 2, 5, and 6; C and D show benzene and toluene, respectively, for Treatments 8, 9, 11, and 12.) Error bars represent 95% confidence intervals.

FIGS. 8A, B, C, and D show BTEX biodegradation—lower concentration—under various anaerobic reducing conditions and in the presence and absence of isobutanol (IBA) or ethanol. (FIG. 8A shows benzene; 8B shows toluene; 8C shows ethylbenzene; 8D shows total xylenes.) Error bars represent 95% confidence intervals.

FIGS. 9 A, B, C, and D show isobutanol biodegradation—lower concentration. (A shows Treatment 3—Unamended; B shows Treatment 7—Nitrate Reducing; C shows Treatment 10—Iron Reducing; D shows Treatment 13—Sulfate Reducing.) Error bars represent 95% confidence intervals. Supplemental monitoring showed that the isobutyric acid concentrations had decreased below the analytical detection limit by day 160 for Treatment 3 and by day 48 for Treatment 13 (data not shown).

FIGS. 10A, B, C, and D show high concentration ethylbenzene and total xylenes biodegradation under various anaerobic reducing conditions and in the presence or absence of isobutanol (IBA). (A and B show ethylbenzene and total xylenes, respectively, for Treatments 1, 2, 5, and 6; C and D show ethylbenzene and total xylenes, respectively, for Treatments 8, 9, 11, and 12.) Error bars represent 95% confidence intervals.

FIGS. 11 and 12 demonstrate the behavior of isobutanol when 1.3% and 2.6% water are initially added to an E10 gasoline. In FIG. 11, increased levels of isobutanol result in reduced aqueous phase volume during phase separation.

FIG. 12 shows there is less ethanol in the aqueous portion when the separation does occur.

FIG. 13 depicts the results from microbial analyses. Error bars represent 95% confidence intervals. IBA=isobutanol.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

In order to further define this invention, the following terms and definitions are herein provided.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers may be added to the specified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. §2111.03.

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

As described herein, hydrocarbon fuel compositions comprising isobutanol provide fuel compositions with increased renewability attributable to a renewable component that is also biodegradable in the environment. Isobutanol is a renewable component which, as shown herein is degraded rapidly when added to aquifer microcosms. Further, results presented herein demonstrate that not only is isobutanol itself biodegraded, but isobutanol also provides additional biodegradation benefits to gasoline under various environmental conditions. As has been described in the art, isobutanol may be biologically produced by microorganisms which convert carbon substrates derived from renewable feedstocks such as biomass into isobutanol. Thus, biologically-produced isobutanol, when added to fuel compositions provides a valuable mechanism for introducing renewable components to fuel and, at the same time, provides for reduced environmental impact of the fuel composition if it were to contaminate a given environmental area.

It is well known that the benzene, toluene, ethylbenzene, and total xylenes (BTEX) components of gasoline are undesirable environmental contaminants. Adding ethanol to gasoline can increase the renewability of the gasoline but this addition may also potentiate the damage resulting from BTEX as ethanol may result in expansion of BTEX plumes. In contrast, adding isobutanol to gasoline provides for a mechanism of increasing renewable component of fuels without increasing a BTEX plume as compared to adding ethanol as the additive should the fuel blend be released an environmental area or compartment (e.g. soil, sediment, groundwater). Further, as shown herein, the addition of isobutanol does not impede BTEX biodegradation to the extent as is observed for ethanol under certain conditions.

“Biodegradation” or “degradation”, as used herein, refers to primary transformation of the compound of interest to a byproduct.

“Improving environmental fate” as used herein means reducing the amount of one or more components of a hydrocarbon fuel blend in an environmental compartment, increasing the degradation rate of one or more components of a hydrocarbon fuel blend in an environmental compartment, decreasing the size of the environmental compartment contacted by one or more components of a hydrocarbon fuel blend, or a combination thereof.

“Environmental compartment” as used herein refers to the area contacted by a fuel composition and may include, for example, soil, sediment, groundwater, or a combination thereof.

“BTEX plume” as used herein refers to a dissolved phase plume.

When used in reference to fuel compositions, “increased renewability” means an increased portion of the composition was produced from resources considered to be renewable, such as biomass, as opposed to resources that are not renewable such as fossil fuels and petroleum.

Provided herein are methods for simultaneously increasing the amount of renewable components in a fuel composition while reducing the environmental impact of a fuel composition by including isobutanol in the fuel composition. The hydrocarbon portion of fuel compositions suitable for the methods disclosed herein comprise gasoline blend stocks useable for making gasolines for consumption in internal combustion engines, including but not limited to spark ignition engines. Gasoline blend stocks include, but are not limited to, blend stocks for gasolines meeting ASTM 4814, EU specification EN228, and blend stocks for reformulated gasoline. Amounts of isobutanol in hydrocarbon fuel compositions (by volume) for the methods disclosed herein include amounts of at least about 2%, at least about 5%, at least at least about 7%, at least about 10%, or at least about 15%. In some aspects, amounts of isobutanol (by volume) include amounts from about 2% to about 20%, and amounts from about 10% to about 16%. It will be appreciated that the amount of isobutanol may be a function of the vehicle technology. As such, in embodiments, isobutanol amounts can be up to about 85% by volume. The isobutanol can be combined with the hydrocarbon portion of the fuel composition using any methods known in the art.

In some embodiments, the hydrocarbon fuel composition further comprises ethanol, and in some embodiments, the ethanol comprises up to about 10%, up to about 15% up to about 20%, or up to about 50% of the hydrocarbon composition prior to the addition of isobutanol. In some embodiments, isobutanol is substituted for ethanol in a fuel composition. In other embodiments, isobutanol is added to a fuel composition which comprises ethanol.

Methods provided herein include a method of retardation of BTEX plume expansion from a hydrocarbon fuel composition by including isobutanol in said composition. The potential environmental impact of a hydrocarbon fuel composition should a release occur can be assessed by measuring the size of BTEX plume in the impacted area, and/or the rate of expansion, and/or the concentration of the BTEX plume. The size and rate of expansion of the BTEX plume can be assessed by methods known in the art such as direct sampling of groundwater and methods such as U.S. Environmental Protection Agency (EPA) method SW846

In some embodiments, degradation of a fuel composition comprising isobutanol occurs faster than a fuel composition comprising ethanol but no isobutanol. Degradation of fuel components can be measured in environmental samples using methods known in the art such as gas chromatography (GC), for example with U.S. EPA method 8015, or GC-mass spectrometry, for example with U.S. EPA method 8260. As shown herein, isobutanol degrades at least as fast as ethanol and, one or more BTEX components degrade faster in the presence of isobutanol than in the presence of ethanol. In some embodiments, isobutanol is degraded with a first order rate constant of at least about 0.081 d⁻¹. In some embodiments, isobutanol is degraded with a first order rate constant of at least about 0.28 d⁻¹. In some embodiments, isobutanol is degraded with a first order rate constant of greater than about 0.074 d⁻¹. In some cases, fuel compositions comprising isobutanol have increased rates of BTEX biodegradation as compared to fuel compositions comprising no renewable component. In some cases, fuel compositions comprising isobutanol have increased rates of BTEX biodegradation as compared to fuel compositions comprising ethanol but no isobutanol.

Furthermore, as shown herein (see FIGS. 11 and 12), incorporating butanol in gasoline containing ethanol reduces the volume of an aqueous phase when phase separation occurs, and limits the weight percent of ethanol contained within the aqueous phase. While not wishing to be bound by theory, it is believed that butanol may limit the amount of ethanol that leaches into groundwater by maintaining it in the less water soluble hydrocarbon fraction. The reduced transport of ethanol in the soil matrix may thereby retard the expansion of BTEX into the water table.

Thus, it will be appreciated, that methods provided herein can advantageously decrease the time needed to remediate a contaminated site, can limit the size of BTEX plumes, or can provide both advantages, therefore improving the environmental fate of a hydrocarbon fuel composition.

Methods provided herein can improve the environmental fate of a hydrocarbon fuel composition comprising isobutanol in an environmental compartment under anaerobic conditions. In embodiments, the methods comprise adding an electron acceptor to said compartment in an amount sufficient to increase the rate of biodegradation of one or more BTEX components. Suitable electron acceptors include nitrates, including, but not limited to NaNO₃, NH₄NO₃, KNO₃, and sulfates, including but not limited to MgSO₄ and CaSO₄, Na₂SO₄, and iron compounds including, but not limited to Fe(OH)₃, chelated iron, zero-valent iron, and nano zero-valent iron. Electron acceptors may be added to an environmental compartment using methods including, but not limited to, injection as a slurry, emplacement, injection through a monitoring well, gravity injection, and/or pressurized injection, or combinations thereof, in amounts sufficient to achieve nitrate-reducing, iron reducing, and/or sulfate reducing conditions. The injection of sulfates and/or nitrates and/or iron compounds may be used to biostimulate sulfate reducing and/or nitrate reducing bacteria, if present, to biodegrade BTEX contamination due the release of isobutanol containing gasoline underground. Such biostimulation may result in increased bioactivity, population, and or metabolism of the bacteria.

Aerobic Conditions

Comparison of isobutanol and ethanol biodegradation rates using a first-order approximation shows that some isobutanol treatments resulted in an observed first-order rate constant of 0.081±0.0044 d⁻¹ (R²=0.80, using data from both Treatment 4 and Treatment 5, Example 1), while ethanol treatment resulted in an observed first-order rate constant of 0.074±0.0023 d⁻¹ (R²=0.90). Thus, isobutanol was biodegraded at a rate greater than that of ethanol in the higher concentration treatments. A rate constant of 0.28±0.054 d⁻¹ (R²=0.69) was attained for isobutanol in the lower concentration treatment (Treatment 3), which is approximately 3.5 times greater than the biodegradation rate constant for either isobutanol or ethanol in the higher concentration treatments. The differences were not due to differences in bacterial growth alone. While not wishing to be bound by theory, it is believed that one explanation for this discrepancy is that isobutanol biodegradation (and growth of isobutanol-degrading bacteria) were nutrient limited over the first two weeks of the study when most of the isobutanol biodegradation occurred. Nutrient limitations may not have been as severe for the lower concentration isobutanol treatment, resulting in an increased rate constant for the lower isobutanol treatment. Alternatively, the discrepancy in isobutanol rate constants may be due to isobutanol substrate inhibition at elevated concentrations. Substrate inhibition was observed during studies of n-butanol biodegradation at concentrations as low as approximately 3,000 μM (Alagappan and Cowan, 2001, Biotechnol and Boengin. 75: 393-405).

Results are presented herein showing that ethanol undesirably reduces the rate of BTEX biodegradation more than isobutanol. Interestingly, benzene exhibited the most pronounced difference when comparing the effects of isobutanol and ethanol on BTEX biodegradation, as benzene aerobic biodegradation was approximately 12-times more slowed in the presence of ethanol than in the presence of isobutanol. Benzene often is the regulatory driver from groundwater contamination at gasoline-contaminated sites.

Anaerobic Conditions

Results presented herein demonstrate that isobutanol is readily biodegraded under nitrate-reducing, iron-reducing, sulfate-reducing, and methanogenic conditions. For the conditions of this study, isobutanol either did not slow BTEX biodegradation, or the extent to which isobutanol lowers the rate of BTEX biodegradation was less than to the extent to which ethanol slowed BTEX biodegradation. In some cases, addition of isobutanol enhanced the observed rates of BTEX biodegradation. Thus, the persistence of isobutanol and its impact on BTEX biodegradation under anaerobic aquifer conditions are considered more favorable than that of ethanol.

EXAMPLES Example 1 Aerobic Testing Materials and Methods

Soil and Groundwater Samples

Soil and groundwater for laboratory microcosm testing were collected from within Site 60 at Vandenberg Air Force Base, CA. The site has a history of gasoline contamination, but has undergone an extensive cleanup program. Collected groundwater was containerized in sterile stainless steel soda kegs (18.5 L) under nitrogen headspace. Soil located approximately 8 to 12 feet below ground surface (bgs) was collected using a Geoprobe® 6620DT with acetate core sleeves. The core samples in acetate sleeves were capped and sealed in the field to minimize exposure to air, shipped overnight on ice to the laboratory, and stored at 4° C.

Soil was removed from the acetate sleeves in an anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, Mich.) and the first 10 cm of the core ends that may have been exposed to oxygen were discarded. Collected soil consisted of silty sand with some gravel and larger stones. The soil was passed through a 0.95 cm sieve, homogenized, and then stored in amber glass jars with Teflon®-lined caps at 4° C. until microcosm setup was complete. Baseline soil and groundwater data are presented in Table 1. (NA=Not Analyzed; * SVOC (semi volatile organic compound) detections include 0.008 mg L⁻¹ phenol and 0.003 mg L⁻¹ bis(2-ethylhexyl) phthalate; ** standard units).

TABLE 1 Baseline soil and groundwater data Parameter Groundwater (mg/L) Soil (mg/kg) Total Organic Carbon  22 1,700 Gasoline Range  5.7 NA Organics Total SVOCs  0.011*   <0.084 Total Iron 490 NA Dissolved Iron  <0.1 NA Nitrate (as N)  1.5 NA Sulfate (as SO₄ ²⁻) 105 NA Alkalinity (as CaCo₃) 391 NA Methane  0.005 NA pH  7.2** NA Dissolved Oxygen  0.8 NA

Microcosm Experiments

The overall approach for the microcosm experiments was to evaluate the biodegradation of BTEX (benzene, toluene, ethylbenzene, and total xylenes) and isobutanol at both “high” and “low” concentrations in soil-groundwater slurries. Final transformation products were not determined. For comparison, one treatment was prepared using ethanol instead of isobutanol. Target BTEX concentrations for each BTEX compound are listed under the BTEX column in Table 2 which shows the experimental treatment matrix. Controls were amended with mercuric chloride and formaldehyde. BTEX and alcohol concentrations were selected to represent (approximately) potential groundwater concentrations that would be observed within a source area and in the near downgradient plume. The greater ethanol concentrations relative to the isobutanol concentrations used in this study were intended to reflect effective solubilities of isobutanol and ethanol in groundwater. Ethanol has an aqueous solubility approximately 10-times that of isobutanol, and the octanol-water partition coefficient of isobutanol is approximately 10-times that of ethanol (Organization for Economic Co-operation and Development, 2004. SIDS Assessment Report for SIAM 19-Ethanol (CAS No. 64-17-5). Berlin, Germany; Organization for Economic Co-operation and Development, 2004. SIDS Assessment Report for SIAM 19-Isobutanol (CAS No. 78-83-1), Berlin, Germany). So an ethanol molar concentration approximately 3-times greater than isobutanol was conservatively selected for the testing in this study. The SET 1 treatments were prepared within 4 days of sample collection whereas SET 2 treatments were prepared after approximately 2 months of soil and groundwater storage. The experimental treatment matrices are shown in Tables 2A and 2B.

TABLE 2A Experimental Treatment Matrix for SET1 BTEX Isobutanol Ethanol Treatment (μM) (μM) (μM) Control 1 180/38/38/75 — — Control 2 180/38/38/75 3,400 — Control 3 15/3.8/3.8/7.5   68 — Treatment 1 15/3.8/3.8/7.5 — — Treatment 2 180/38/38/75 — — Treatment 3 15/3.8/3.8/7.5   68 — Treatment 4 180/38/38/75 3,400 —

TABLE 2B Experimentai Treatment Matrix for SET2 BTEX Isobutanol Ethanol Treatment (μM) (μM) (μM) Control 4 180/38/38/75 — 11,000 Treatment 5 180/38/38/75 3,400 — Treatment 6 180/38/38/75 — 11,000

Microcosms were prepared by placing 40 g of site soil into each of 54 glass serum bottles (approximate volume 160 mL each). BTEX and alcohol (isobutanol or ethanol) were added to the treatment bottles to attain the target concentrations shown in Tables 2A and B. Bottles were filled with groundwater so as to leave 10 mL of headspace. Controls were amended with mercuric chloride (700 mg/L in bottles) to inhibit microbial activity. Controls were subsequently amended with formaldehyde (1% v/v in bottles) after 4 days to limit microbial activity. Treatments were prepared with a minimum of 3 and up to 8 replicates each.

The prepared microcosms were incubated at 15° C. on a rotary shaker operating at 100 rpm. Headspace in each of the bottles was monitored for BTEX and oxygen. Aqueous BTEX concentrations were calculated by applying Henry's Law. Samples of the aqueous phase were analyzed for isobutanol and ethanol, as well as potential isobutanol degradation products (iso-butylaldehyde and iso-butyric acid). The headspace of each bottle was periodically flushed with oxygen gas to maintain aerobic conditions in the bottles. The headspace of each control bottle also was flushed with oxygen to evaluate potential losses of BTEX due to the flushing process.

Analytical Methods

Headspace gases were analyzed for BTEX by using a Varian CP-3900 gas chromatograph equipped with an FID and a Restek QSPLOT column (30 m length, 0.32 mm diameter) with an injector temperature of 260° C. and a detector temperature of 290° C. Headspace oxygen was analyzed by using a Varian CP-3800 gas chromatograph equipped with a Pulsed Discharge Helium Ionization Detector (PDHID) and both a CP-Molsiene 5A column and a CP-ParaBond Q column (both 10 m length, 0.32 mm diameter), an injector temperature of 210° C., and a detector temperature of 240° C. Oxygen eluted at 1.20 min. Aqueous alcohol concentrations (as well as iso-butylaldehyde and iso-butyric acid) were measured by first collecting a 130 μL subsample preserved with mercuric chloride. These samples were analyzed by using a Varian CP-3800 gas chromatograph equipped with a flame ionization detector (FID) and a Stabilwax DA column (30 m length, 320 μm diameter), an injector temperature of 280° C., and a detector temperature of 280° C.

Microbial Analyses

Samples (2 mL) were collected from microcosms amended with BTEX and isobutanol at the start, midway (approximately), and end of the experiment to evaluate changes in the microbial population over the course of the study. Samples were serially diluted and plated onto R2A Agar (BD Difco) and Basal Salts Medium (BSM; Hareland et al., 1975, J. Bacteriol. 121: 272-285.) agar immediately after retrieval as per Method SM9215C. BSM plates were incubated in sealed containers with either BTEX or isobutanol to select for bacteria capable of growth on these substrates. Colony counts were performed manually after 3 days for R2A agar and after 10 days for selective media. Samples (˜8 mL) also were immediately frozen at −70° C. and at the conclusion of the study were shipped on dry ice to Microbial Insights, Rockford, Tenn., for CENSUS analysis. CENSUS employed quantitative polymerase chain reaction (qPCR) assays to quantify total Eubacteria based on enumeration of Eubacterial 16S rRNA gene copies (for more information on the CENSUS method see Microbial Insights, 2009http://www.microbe.com/how-census-works.html and http://microbe.com/census-applications/anaerobic-btex.html; accessed Jul. 7, 2009 and May 8, 2009, respectively).

Isobutanol and Ethanol Degradation

No measurable decreases (>10%) in isobutanol or ethanol, accumulation of isobutylaldehyde, or accumulation of isobutyric acid were observed in any of the controls. Isobutanol biodegradation in Treatments 3 and 4 are shown in FIG. 1. In the lower concentration treatment (Treatment 3), isobutanol was degraded to below the analytical detection limit (3 μM) within 7 days. Isobutanol degradation products isobutylaldehyde and isobutyric acid were first detected at 4 days of incubation, with isobutylaldehyde subsequently decreasing to below detection within 5 days. Isobutyric acid concentrations increased initially, but samples taken after 82 days confirm that isobutyric acid also was further degraded in the microcosms (data not shown). The biodegradation products of isobutyric acid were not determined, but previous studies have shown that isobutyric acid readily biodegraded under aerobic conditions, and that butyrate is readily transformed to CO₂ under aerobic conditions (Miller, 2001, J. Anim. Sci. 79: 2503-2512; Bonartseva, 2003, Appl. Biochem. Biotech. 109: 285-301).

In the higher concentration treatment (Treatment 4, FIG. 1 b), isobutanol was degraded from an initial concentration of 3,400 μM to below the analytical detection limit within 23 days. As isobutanol was degraded, the formation and subsequent degradation of two isobutanol breakdown products, isobutylaldehyde and isobutyric acid, were observed. Isobutylaldehyde reached a peak concentration of 900 μM on day 9 and then declined below the analytical detection limit of 11 μM after 19 days. Isobutyric acid increased to a peak concentration of 1,750 μM on day 25 and then decreased to 100 μM by day 48. In the second set of microcosms used for comparing isobutanol and ethanol (Treatment 5), isobutanol was degraded in a similar timeframe and isobutyric acid was observed in similar quantities. However, only trace levels of isobutylaldehyde (78 μM) were observed.

In ethanol amended microcosms, ethanol concentrations decreased from 11,000 μM to below the analytical detection limit of 22 μM in approximately 40 to 45 days (FIG. 1 c). No decreases in ethanol were observed in the controls. To ensure that the observed ethanol degradation was not limited by available nutrients, nutrients in the form of a modified basal salt media (Hareland et al., 1975) were added to all treatments on day 22. No measurable increase in the rate of ethanol (or any other compound) degradation was observed, suggesting that contaminant biodegradation was not limited by nutrient availability near day 22 in the experimental system.

BTEX Degradation

Microcosms amended with higher concentrations of BTEX, with and without isobutanol, are shown in FIG. 2. Results for the lower concentration BTEX are provided in FIG. 4. Observed lag times and regressed first-order effective half lives are in days and shown in Table 3. Effective half-lives are the regressed half-life plus the lag time. (IBA=isobutanol; ± values indicate 95% confidence intervals.)

TABLE 3 Lag times and regressed effective half-lives (τ_(1/2)) for BTEX. Benzene Toluene Ethylbenzene Total Xylenes Treatment Lag τ_(1/2) Lag τ_(1/2) Lag τ_(1/2) Lag τ_(1/2) 1 (Low BTEX) 0 2.0 ± 0.33 0 1.4 ± 0.17 2 2.8 ± 0.08 2 2.8 ± 0.06 2 (High BTEX) 6 7.7 ± 0.34 5 5.8 ± 0.08 5 5.9 ± 0.11 0 4.1 ± 0.33 3 (Low BTEX + IBA) 0 2.7 ± 0.33 0 1.6 ± 0.18 1 1.8 ± 0.10 1 2.0 ± 0.08 4 (High BTEX + IBA) 1 13 ± 1.8  5 8.3 ± 0.31 0 4.1 ± 0.39 0 6.9 ± .57  5 (High BTEX + IBA) 1 2.4 ± .09  1 2.3 ± 0.10 1 1.9 ± 0.07 1 4.8 ± 0.33 6 (High BTEX + Ethanol) 1 30 ± 4.3  1 9.3 ± 0.96 1 1.7 ± 0.05 1 22 ± 3.6 

BTEX concentrations in the high concentration controls showed no observable decreasing trend through approximately 25 days, at which time decreases in the control concentrations were observed for some compounds (up to approximately 20%). The controls were subsequently re-amended with formaldehyde to inhibit microbial activity; of the additional formaldehyde and to prevent further decreases in the controls. By 25 days, most of the BTEX compounds had been degraded, so these losses did not impact evaluation of the data. No significant (>10%) decreases in the low concentration BTEX controls were observed, except for the total xylenes where an approximately 25% decrease in total xylenes concentration was observed over the 10-day duration of this experiment.

Half lives obtained for the BTEX compounds under aerobic conditions (Table 3) were generally within the range observed by others (see review in United States Geological Survey, 2006, “Description, properties, and degradation of selected volatile organic compounds detected in groundwater—a review of selected literature. Open File Report 2006-1338). Results for the comparison microcosms (Treatments 4, 5 and 6) show that ethanol generally exhibited greater adverse impacts on BTEX biodegradation than isobutanol did (FIG. 3 and Table 3). The only exception was ethylbenzene, where isobutanol and ethanol impacted ethylbenzene biodegradation similarly. Moreover, the addition of isobutanol resulted in a small but measurable increase in ethyl benzene and total xylenes biodegradation in the low concentration treatments (Treatments 1 and 3), possibly due to the fortuitous growth of ethylbenzene and total xylene-degraders on isobutanol.

The effective half lives for BTEX in Treatment 5 were less than in Treatment 4 (Table 3) by approximately a factor of two to five. However, the rate of isobutanol biodegradation in these two treatments was approximately the same.

Microbial Characterization.

Results of microbial colony counts and qPCR analyses are shown in FIG. 13. These data show that microbial concentrations in the microcosms increased throughout the duration of the study. Both the microbial colony counts and total Eubacteria data show similar trends, although Eubacteria concentrations determined by molecular analysis were greater than the microbial colony counts. A discrepancy between aerobic plate counts and total bacterial by molecular analysis is not unusual because some bacteria do not grow well on all agar plates, and the latter method measures culturable, non-culturable, anaerobic, and aerobic bacteria. The plate count data confirmed that natural bacteria capable of degrading both isobutanol and BTEX were present in the site materials. As indicated in the low concentration treatments, microbial growth became limited when substrate (either BTEX or isobutanol) was depleted.

Example 2 Anaerobic Testing Materials and Methods Soil and Groundwater

The soil and groundwater and sample collection and handling procedures are as described above. Baseline soil and groundwater data are presented in Table 4. (NA=Not Analyzed; * SVOC detections include 0.008 mg L-1 phenol and 0.003 mg L-1 bis(2-ethylhexyl) phthalate; ** standard units)

TABLE 4 Groundwater and soil parameters Parameter Groundwater (mg/L) Soil (mg/kg) Total Organic Carbon  22 1,700 Gasoline Range  5.7 NA Organics Total SVOCs  0.011*   0.084 Total Iron 490 NA Dissolved Iron  <0.1 NA Nitrate (as N)  1.5 NA Sulfate (as SO₄ ²⁻) 105 NA Alkalinity (as CaCo₃) 391 NA Methane  0.005 NA pH  7.2** NA Dissolved Oxygen  0.8 NA

Microcosm Preparation

For the microcosm experiments, the biodegradation of BTEX and isobutanol in soil-groundwater slurries under conditions ranging from nitrate-reducing to methanogenic were evaluated. BTEX and isobutanol were evaluated at “high” and “low” concentrations (Table 5). Two treatments were prepared using ethanol instead of isobutanol. Electron acceptor concentrations reflect the amount added to the sample and (Table 5) do not include background electron acceptor concentrations in site groundwater. BTEX and alcohol concentrations represent potential groundwater concentrations that might be observed within a source area and in the near downgradient plume. The greater ethanol concentrations relative to the isobutanol concentrations used in this study were intended to reflect effective solubilities of isobutanol and ethanol in groundwater. Ethanol has an aqueous solubility approximately 10-times that of isobutanol, and the octanol-water partition coefficient of isobutanol is approximately 10-times that of ethanol (Organization for Economic Co-operation and Development, 2004. SIDS Assessment Report for SIAM 19-Ethanol (CAS No. 64-17-5). Berlin, Germany; Organization for Economic Co-operation and Development, 2004. SIDS Assessment Report for SIAM 19-Isobutanol (CAS No. 78-83-1), Berlin, Germany). Because the rate of ethanol biodegradation was anticipated to be greater than that of isobutanol, an ethanol molar concentration approximately 3-times greater than isobutanol was conservatively selected for testing.

All microcosm preparation was performed in an anaerobic chamber. Microcosms were prepared by placing 40 g of site soil into 160 mL glass serum bottles. BTEX and alcohol were added to the treatment bottles to attain the target concentrations shown in Table 5. (Target BTEX concentrations for each BTEX compound are listed under the BTEX column.) Bottles were filled with groundwater, leaving approximately 2 mL of headspace. Control microcosms were amended with mercuric chloride (700 mg L⁻¹ in bottles) and formaldehyde (1% by volume in bottles) to limit microbial activity. Treatments were prepared with a minimum of 3 replicates for alcohol and BTEX analyses; one additional replicate per treatment was used for monitoring electron acceptors and methane.

The bottles were crimp sealed with Teflon-lined butyl stoppers, and incubated at 15° C. on a rotary shaker operating at 100 rpm. If needed, additional electron acceptor was added to maintain the desired reducing conditions. Controls were amended with mercuric chloride and formaldehyde. Amended electron acceptor concentrations are shown in the last column. Nutrients, in the form of a modified basal salt medium (Hareland et al., 1975), were added to all treatments at 22 days.

TABLE 5 Experimental Treatment Matrix BTEX Isobutanol Ethanol Electron Treatment (μM) (μM) (μM) Acceptor (μM) Control 1 180/38/38/75 — — — Control 2 180/38/38/75 3,400 — — Control 3 15/3.8/3.8/7.5   68 — — Control 4 15/3.8/3.8/7.5 — 11,000 — Treatment 1 180/38/38/75 — — — Treatment 2 180/38/38/75 3,400 — — Treatment 3 15/3.8/3.8/7.5   68 — — Treatment 4 15/3.8/3.8/7.5 — 11,000 — Treatment 5 180/38/38/75 — — 7,100 (NaNO₃) Treatment 6 180/38/38/75 3,400 — 7,100 (NaNO₃) Treatment 7 15/3.8/3.8/7.5   68 — 7,100 (NaNO₃) Treatment 8 180/38/38/75 — — 2,600 (Fe(OH)₃) Treatment 9 180/38/38/75 3,400 — 2,600 (Fe(OH)₃) Treatment 10 15/3.8/3.8/7.5   68 — 2,600 (Fe(OH)₃) Treatment 11 180/38/38/75 — — 3,200 (MgSO₄) Treatment 12 180/38/38/75 3,400 — 3,200 (MgSO₄) Treatment 13 15/3.8/3.8/7.5   68 — 3,200 (MgSO₄) Treatment 14 15/3.8/3.8/7.5 — 11,000 3,200 (MgSO₄)

Analytical Methods

Headspace gases were analyzed for BTEX by using a Varian CP-3900 gas chromatograph, (GC) equipped with a flame ionization detector (FID) and a Restek QSPLOT column, and for methane by using a GC equipped with an FID and a Restek Rt-Alumina column Aqueous concentrations were calculated by applying Henry's Law.

Aqueous alcohol concentrations (as well as potential isobutanol degradation products iso-butylaldehyde and iso-butyric acid) were analyzed by using a Varian CP-3800 gas chromatograph, equipped with an FID and a Stabilwax DA column. Aqueous samples were collected for anions analysis via ion chromatography (Dionex DX-120, Sunnyvale, Calif.). Nitrate also was periodically measured using Quant Nitrate Test strips (EMD Chemicals, Gibbstown, N.J.). Total and dissolved iron were measured using Hach test Kits (Hach, Loveland, Colo.) according to the manufacturer's instructions.

Microbial Analyses

Microcosm slurry sub-samples samples (approximately 8 mL) were obtained from Treatments 2, 6, 9, and 12 at the start and at the end of the experiment to ascertain changes in the microbial population over the course of the study. Samples were immediately frozen at −70° C. and (at the conclusion of the study) were shipped on dry ice to Microbial Insights, Rockford, Tenn., for quantitative polymerase chain reaction (qPCR) of total Eu bacteria, denitrifying bacteria, iron and sulfate reducing bacteria, and methanogenic bacteria by the CENSUS™ quantitative PCR technique (Microbial Insights, 2009http://www.microbe.com/how-census-works.html and http://microbe.com/census-applications/anaerobic-btex.html; accessed Jul. 7, 2009 and May 8, 2009, respectively).

Isobutanol and Ethanol Degradation

Isobutanol was completely degraded in the high concentration treatments, and its degradation rates varied under different anaerobic conditions. FIG. 5 shows isobutanol and electron acceptor concentrations in the higher concentration treatments (Treatments 2, 6, 9, and 12). Background sulfate concentrations are shown for the unamended treatment. No decreases in isobutanol or ethanol, nor accumulation of isobutylaldehyde or isobutyric acid, were observed in the control microcosms. The most rapid isobutanol biodegradation was observed in the nitrate-amended microcosms. Within 16 days, isobutanol was degraded to below detection limits under nitrate-reducing conditions (Treatment 6). Nitrate was utilized concurrently with the isobutanol degradation, and decreased to non-detect by day 13 before being re-amended on day 14. No measurable decrease in the background sulfate was observed in Treatment 6 through 19 days (data not shown).

Isobutanol biodegradation was observed in both the sulfate amended treatments (Treatment 12) and the unamended microcosms (Treatment 2) where limited background sulfate existed. To evaluate biodegradation of isobutanol after depletion of sulfate (i.e., methanogenic conditions), microcosms bottles for Treatment 2 were re-spiked with isobutanol (to a final concentration of 3,400 μM) at 88 days. The additional isobutanol was degraded within approximately 30 days. However, only trace (<2 μM) levels of methane were observed in Treatment 2 following depletion of the sulfate, which is similar to the methane levels in the controls.

Isobutanol in the iron-amended treatment was biodegraded to below the analytical detection limit within approximately 80 days. Ferric iron, monitored through 44 days, showed concentrations ranging from approximately 18 to 36 μM. However, only relatively low levels of dissolved ferrous iron (up to 36 μM) were observed. The absence of appreciable ferrous iron accumulation was likely the result of iron sulfide formation in the microcosm bottles, as a black precipitate was observed. In addition, and consistent with the formation of iron sulfides, background sulfate concentrations decreased in the iron-amended treatment. Decreases in isobutanol concentrations correlated with decreases in sulfate levels.

In the lower isobutanol concentration microcosms (Treatments 3, 7, 10, and 13) isobutanol was completely biodegraded within approximately 25 days in the iron-amended treatment, and approximately 15 days in the unamended, nitrate-amended and sulfate-amended treatments (FIG. 9). In addition to the amended electron acceptors, each microcosm contained approximately 100 μM background nitrate levels in the groundwater material, and background nitrate decreases correlating with isobutanol biodegradation were observed during the incubation. The degradation of one mole of isobutanol theoretically requires four moles of nitrate which is assumed to be completely reduced into nitrogen (McCarty, “Bioengineering issues related to in situ remediation of contaminated soils and groundwater” Environmental Technology, in: Omenn, G. S., Reducing risks from environmental chemicals through biotechnology. Plenum press, New York, pp. 143-162, 1988). Enrichment culture experiments suggested that the mole ratio of nitrate consumed and benzene degraded was ten, twice the theoretical number (Burland and Edwards, 1999, Appl Environ Microbiol 65(2): 529-533). The 100 μM background nitrate could facilitate the biodegradation of approximately 13 μM isobutanol, 18% of the initial isobutanol in low concentration treatments. No background sulfate reduction was observed in the low concentration treatments.

Isobutyric acid and trace levels of isobutylaldehyde were identified as transient biodegradation intermediates; the subsequent biodegradation of both of these compounds was observed in all treatments. Iso-butyric acid accumulated to near (with a factor of approximately 2) stoichiometric quantities, with the exception of the high concentration nitrate-amended treatment in which only 5% accumulation was observed. The limited generation of isobutyric acid in the high concentration nitrate treatment is not readily explained.

In ethanol-amended microcosms (Treatments 4 and 14), ethanol was degraded to below the analytical detection limit in approximately 60 days under sulfate reducing conditions (FIG. 6). Methane was detected in both Treatments 4 and 14 (4 and 25 μM, respectively) at day 44, but decreased to non-detect levels by day 78.

To evaluate ethanol biodegradation after depletion of sulfate, ethanol was re-spiked to Treatment 4 bottles at 88 days. Subsequent biodegradation of the ethanol to below the analytical detection limit occurred within 90 days. This result is consistent with previous anaerobic ethanol studies documenting ethanol biodegradation via fermentation in the absence of sulfate (Laanbroek et al., 1982, Arch. Microbial. 133: 178-184).

BTEX Degradation.

Decreases in BTEX concentrations in the controls were negligible (<15%) during the course of the experiments. BTEX biodegradation in the higher concentration treatments are shown in FIGS. 5 and 7, lower concentration BTEX results (with comparative ethanol treatments) are presented in FIG. 8. No data are available for the last two time points for Treatment 4 because BTEX was inadvertently re-spiked into this treatment.

Toluene biodegradation was observed in all the high concentration microcosms amended with electron acceptors (FIG. 7). When incubated without alcohols, approximately 380 toluene was degraded to below detection limits within 80 days under nitrate-reducing, iron-reducing and sulfate-reducing conditions, respectively. The presence of isobutanol exhibited slight impacts on toluene degradation under nitrate-reducing and sulfate-reducing conditions, but slowed the toluene degradation in the iron amended microcosms. In the unamended high concentration microcosms (Treatment 2), however, no measurable toluene concentration decreasing trend relative to the controls was observed. Background sulfate in Treatment 2 became depleted in the first 30 days, presumably due to the isobutanol biodegradation, and the lack of electron acceptors was likely the reason of toluene persistence in the unamended treatments. In the lower concentration microcosms where sufficient electron acceptors were present, isobutanol exhibited little impacts on the toluene biodegradation in both the unamended (limited sulfate) and any of the microcosms amended with electron acceptors (FIG. 8). Results for ethylbenzene and total xylenes (FIG. 10) were similar to those for toluene, although biodegradations of ethylbenzene and total xylenes occurred slightly slower compared to toluene.

In the higher BTEX concentration treatments without isobutanol, no substantial benzene biodegradation was observed under any anaerobic conditions throughout 162 days of incubation (FIG. 7). However, the presence of isobutanol appeared to stimulate benzene biodegradation under iron-reducing and sulfate-reducing conditions, as the benzene concentrations in Treatments 9 and 12 started to decrease by the end of the experiment. In the low concentration treatments under sulfate-reducing conditions, benzene concentrations decreased to below the analytical detection limit (0.50 μM) in the microcosms with isobutanol (Treatment 13), and to approximately 0.85 μM in the microcosms with ethanol (Treatment 14) after approximately 300 days of incubation. Benzene biodegradation started before day 120 in Treatment 14, and occurred between 160 and 300 days in Treatment 13.

Microbial Analyses

Results of the microbial analyses, shown in Table 6, generally showed an increase in microbial concentrations during the incubations. (Values are in cells mL⁻¹; ± values indicate 95% confidence intervals.)

TABLE 6 Results of microbial analyses Denitrifying Denitrifying Iron & Sulfate- Total bacteria bacteria reducing bacteria Methanogens Treatment Days Eubacteria (nirS) (nirK) (δ-protobacteria) (mcrA) Tr. 2- 0 8.2 ± 7.7 × 10⁸ 1.0 ± 0.8 × 10⁹ 5.9 ± 7.9 × 10⁵ 1.3 ± 0.4 × 10⁴ 5.4 ± 4.7 × 10⁵ Unamended 132 5.2 ± 4.1 × 10⁸ 1.3 ± 0.5 × 10⁹ 1.9 ± 1.7 × 10⁷ 1.4 ± 0.3 × 10⁷ 3.8 ± 2.9 × 10⁷ Tr. 6- 0 5.6 ± 4.5 × 10⁷ 3.3 ± 5.8 × 10⁸ 1.0 ± 0.9 × 10⁶ 2.6 ± 3.0 × 10³ 4.5 ± 3.0 × 10⁵ Nitrate 14 5.0 ± 4.9 × 10⁸ 5.9 ± 5.3 × 10⁸ 5.1 ± 5.6 × 10⁶ 2.5 ± 1.2 × 10³ 4.7 ± 3.3 × 10⁴ reducing 132 1.3 ± 0.5 × 10⁹ 1.3 ± 0.2 × 10⁹ 5.6 ± 4.7 × 10⁷ 0.8 ± 1.1 × 10⁴ 4.6 ± 4.3 × 10⁷ Tr. 9-Iron 0 8.1 ± 2.8 × 10⁷ 4.6 ± 4.0 × 10⁸ 1.2 ± 0.6 × 10⁶ 2.0 ± 3.5 × 10⁴ 2.5 ± 1.0 × 10⁵ reducing 132 1.5 ± 1.5 × 10⁸ 5.6 ± 4.0 × 10⁸ 1.9 ± 0.4 × 10⁷ 1.7 ± 1.9 × 10⁷ 1.9 ± 1.9 × 10⁷ Tr. 12- 0 6.0 ± 0.9 × 10⁷ 2.7 ± 3.9 × 10⁸ 3.9 ± 1.5 × 10⁵ 2.2 ± 2.4 × 10⁴ 1.1 ± 0.4 × 10⁶ Sulfate 132 7.1 ± 7.0 × 10⁸ 1.3 ± 0.5 × 10⁹ 6.7 ± 3.3 × 10⁶ 1.1 ± 0.3 × 10⁷ 3.1 ± 1.9 × 10⁷ reducing

In the microcosms containing ferric iron (Treatment 9) or sulfate (Treatments 2, and 12), the population of corresponding iron reducing and sulfate reducing bacteria increased 1000-fold, respectively. As nitrate was depleted toward the end of the anaerobic incubation in Treatment 6, the methanogen population increased 100-fold. Slightly lower levels of methanogen biomass increase were observed in the other treatments.

Increased growth of denitrifying bacteria was not observed in the nitrate amended treatments. One explanation for this observation is that, although two different nitrate reductase genes were monitored, the functional genes for the denitrifying bacteria in our system were not quantified. Alternately, while the activity of the denitrifying bacteria may have been substantial, growth of denitrifying bacteria may have been low, as observed in n-butanol studies under denitrifying conditions by Dubbels et al. (2009, Int. J. Syst. Evol. Microbiol. 59: 1576-1578). Likewise, fermentative bacteria that do not rely on nitrate reduction for growth also may be involved in isobutanol degradation under denitrifying conditions (Laanbroek et al., 1982, Arch. Microbiol. 133: 178-184).

Example 3 Isobutanol as a Co-Solvent for Ethanol

Scoping water tolerance and phase separation tests were conducted on isobutanol-ethanol-gasoline blends at 65° F. Increasing amounts of isobutanol were mixed with E10 gasoline and then either 1.3% or 2.6% water were added to all of the blends. The water needed to be increased to 2.6 vol % for some blends because gasoline blends containing ethanol absorb larger amounts of water and 1.3% water was not always sufficient to induce the formation of separate aqueous and hydrocarbon phases for analysis (i.e., the 1.3 vol % water was completely absorbed by the higher isobutanol blends). This can be seen in FIG. 11 where 1.3 vol % was sufficient to cause two phases until the amount of isobutanol in E10 reached 5 vol %, but for 10 vol % isobutanol in E10, the level needed to be increased to 2.6 vol % water to induce phase separation.

The data in FIG. 12 show that the amount of ethanol extracted into the aqueous phase decreased with increasing iso-butanol concentration indicating that iso-butanol acts as a co-solvent for ethanol. Concentrations in both the hydrocarbon and aqueous phase were determined by GC.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. 

1. A method for increasing the renewability of a hydrocarbon fuel composition and limiting the impact on an environmental compartment upon contamination by said hydrocarbon fuel composition, comprising adding a suitable amount of isobutanol in said hydrocarbon fuel composition for increasing the biodegradability of the hydrocarbon fuel composition.
 2. The method of claim 1, wherein the hydrocarbon fuel composition further comprises ethanol.
 3. The method of claim 1, wherein the ethanol comprises up to about 10% of the fuel composition prior to addition of isobutanol.
 4. The method of claim 1, wherein the isobutanol provides for improved biodegradation of at least one of the BTEX components of the hydrocarbon fuel composition.
 5. The method of claim 1 wherein the isobutanol provides for improved biodegradation of benzene.
 6. The method of claim 2, wherein the environmental compartment includes a soil matrix and wherein the addition of isobutanol reduces the transport of ethanol in a soil matrix.
 7. The method of claim 1 wherein the addition of isobutanol impedes expansion of a BTEX plume from said composition.
 8. The method of claim 1, wherein the addition of isobutanol enhances the biodegradability of various components of the hydrocarbon fuel composition.
 9. The method of either of claim 4 or 5 wherein the improved biodegradation occurs under aerobic conditions.
 10. The method of either of claim 4 or 5 wherein the improved biodegradation occurs under nitrate-reducing or sulfate-reducing conditions.
 11. A method of improving the environmental fate of a hydrocarbon fuel composition comprising isobutanol in an environmental compartment under anaerobic conditions comprising adding an electron acceptor to said compartment in an amount sufficient to increase the rate of biodegradation of one or more BTEX components.
 12. The method of claim 11 wherein the electron acceptor is iron, sulfate, or nitrate, or a combination thereof.
 13. The method of claim 11 wherein the electron acceptor is Fe(OH)₃.
 14. The method of claim 11 wherein the electron acceptor is NaNO₃.
 15. The method of claim 11 wherein the electron acceptor is MgSO₄.
 16. The method of claim 11 wherein the one or more BTEX components comprise toluene.
 17. The method of claim 11 wherein the one or more BTEX components comprise xylene.
 18. The method of claim 11 wherein the one or more BTEX components comprise benzene.
 19. The method of claim 11 wherein the electron acceptor is nitrate and is added in an amount sufficient to create nitrate-reducing conditions.
 20. The method of claim 11 wherein the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions.
 21. The method of claim 11 wherein the electron acceptor is nitrate and is present in an amount sufficient to create nitrate-reducing conditions and wherein toluene biodegrades in about the same number of days as isobutanol.
 22. The method of claim 11 wherein the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein toluene biodegrades in about the same number of days as isobutanol.
 23. The method of claim 11 wherein the electron acceptor is nitrate and is present in an amount sufficient to create nitrate-reducing conditions and wherein benzene biodegrades in about the same number of days as isobutanol.
 24. The method of claim 11 wherein the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation without sulfate-reducing conditions.
 25. The method of claim 11 wherein the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation in the absence of isobutanol.
 26. The method of claim 11 wherein the electron acceptor is sulfate and is present in an amount sufficient to create sulfate-reducing conditions and wherein benzene biodegradation is improved as compared to its biodegradation in the presence of ethanol.
 27. A composition comprising gasoline, isobutanol and at least one of Fe(OH)₃, NaNO₃, or MgSO₄.
 28. A composition comprising gasoline, isobutanol and at least one of Fe(OH)₃, NaNO₃, KNO₃, NHNO₃, Na₂SO₄, CaSO₄, MgSO₄, chelated iron, zero-valent iron, and nano zero-valent iron. 